A useful modality for analyzing many types of samples is the measurement of luminescence (emission of light) by the sample. For example, in the case of a biological sample, luminescence is useful in the study of biochemical reactions, cellular physiology, gene expression, etc. Luminescence in a sample may result from the activity of a type of reporter enzyme known as luciferase, along with the injection of another reagent, coenzyme, and/or catalyst as needed for a particular application. Luminescence measurement may involve the use of a single type of luciferase. However, a so-called dual-reporter assay that entails the use of two different types of luciferase, such as firefly luciferase and Renilla luciferase, is often desired for its ability to minimize experimental variability and thereby improve the reliability of the data acquired. The second type of luciferase generates a subsequent signal while a quenching reagent suppresses or extinguishes the signal produced by the reaction with the first type of luciferase.
In an example of a dual-reporter assay protocol, the first type of luciferase is present in the pre-injection sample and the reagent generating the first signal is then added to the sample. After a delay period of typically a few seconds (e.g., two seconds), the resulting luminescent activity is measured over a period of time such as ten seconds. Then the reagent generating the second signal from the second type of luciferase is dispensed into the sample along with the quenching reagent. After a second delay period of again typically a few seconds, the resulting luminescent activity is measured over a period of time such as ten seconds. This dual-reporter assay protocol may be performed using a single-tube luminometer or a microplate (multi-well optical reader plate) luminometer.
A significant aspect of the dual-reporter assay is the mixing of the quenching reagent with the sample. The mixing needs to be effective enough, over the time period allotted by the protocol for mixing (after reagent injection and prior to measuring the activity of the second luciferase), to provide effective quenching of the signal produced by the first luciferase, and thereby to allow separation of the second signal from the first signal and thus acquire high-quality data. For example, the protocol may call for the first signal to be suppressed by 4 logs (more than 10,000 fold) before measuring the second signal. The afore-mentioned delay (mixing) period of about two seconds is often adequate for effective mixing when operating a single-tube or microplate luminometer with a conventional, relatively high reagent injection speed. A high injection speed, for example a flow rate on the order of hundreds of microliters per second (μL/sec), often imparts enough turbidity in the sample tube or well to result in effective mixing over a short period of time. However, for many microplate applications a lower injection speed would be desirable for reasons such as ensuring high dispensing accuracy, for example down to 1-μL increments. Due to the lower turbidity imparted by lower injection speeds, a short delay period may not provide sufficient time for effective mixing and consequently may not provide sufficient time for adequate quenching. Therefore, there is a need for providing a way to enhance or accelerate mixing in certain applications such as those utilizing low injection speeds and/or constrained to short mixing periods.
Moreover, when performing assays on a large number of samples using microplates, taking luminescence measurements on each sample over a long integration time such as ten seconds can require a large amount of total plate read time. Accelerating mixing may enable a reduction in the amount of time needed for taking luminescence measurements on each sample. Therefore, there is a need for providing a way to enhance or accelerate mixing in applications for which higher throughput is desired.